Purification, characterization, and cDNA cloning of a novel metallothionein-like, cadmium-binding protein from Caenorhabditis elegans.
Caenorhabditis elegans adapted for survival in high concentrations of Cd(II) express a heavy metal binding protein designated C. elegans metallothionein-like protein or MT-Ce. This protein was purified to homogeneity and characterized. MT-Ce binds 6 mol of Cd(II)/mol protein. The sequence of 39 amino-terminal residues in MT-Ce was determined. A radiolabeled 41-mer oligonucleotide, designed from the partial MT-Ce sequence, was used in conjunction with sucrose gradient centrifugation to obtain size-fractionated poly(A+) RNA enriched in MT-Ce sequences. Subsequently, cloned cDNAs, corresponding to MT-Ce mRNA sequences, were isolated from a lambda ZapII cDNA library prepared from the enriched template mRNA. cDNA and protein sequence analysis revealed that MT-Ce comprises 62 amino acid residues and has a predicted Mr of 6462. Seventeen of the 18 Cys residues in the nematode cadmium-binding protein are included in Cys-X-Cys and X-Cys-Cys-X motifs that are characteristic of mammalian metallothioneins (MTs). However, the resemblance of MT-Ce to mammalian MTs is superficial. The amino acid sequence of MT-Ce is unique, and neither its putative alpha and beta domains nor its Cys residues can be readily aligned with the corresponding regions of other eukaryotic MTs. This suggests that MT-Ce is an example of convergent evolution. The MT-Ce mRNA level in nematodes that were selected and grown with Cd(II) concentrations that are lethal for wild-type worms, was 55-fold higher than the level of MT-Ce mRNA in wild-type C. elegans. Comparison of the sequences of MT-Ce cDNAs revealed the occurrence of two types of MT-Ce mRNA. Each contains an identical coding region, but the cDNAs diverge markedly in their 5'-untranslated regions. This suggests the possibilities of regulation by alternative splicing and/or the presence of multiple MT-Ce genes encoding a single protein, but controlled by different regulatory elements.
Slice
L W
LW
Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461.
Freedman
J H
JH
Rubin
C S
CS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002104:Cadmium / Q000378:metabolism; D002107:Caenorhabditis / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D008668:Metallothionein / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008670:Metals / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet
D008670:Metals; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; C004203:cadmium-binding protein; D002104:Cadmium; D004247:DNA; D008668:Metallothionein